Preparation and Culture of Protoplasts of some. Japanese Cultivated Mushrooms

Transcription

1 #l5l:lilf it Bull. For. & For. Prod. Res. Inst. No. 343, 1987 Preparation and Culture of Protoplasts of some Japanese Cultivated Mushrooms By Masatake OHMAsAm, Yasuhisa ABE'2>, Hisahiko FuRUKAWA's> Minoru TANIGUCHIW and Hitoshi NEnAcs> Summary : Protoplasts were prepared from mycelia of 12 mushrooms, Lentinus edodes, Pleurotus ostreatus, Flammulina velutipes, Pholiota nameko, Grifola frondosa, Tricholoma matsutake and others. Mycelia of these mushrooms were cultured in liquid media, collected by filtration and treated with mixtures of commercial mycolytic enzymes. Among combinations of enzymes, enzyme systems containing a) Cellulase "Onozuka'' RS and,8-giucronidase, b) Cellulase "Onozuka" RS, Driselase and,8-glucronidase and c) Cellulase "Onozuka" RS, Driselase and Zymolyase 5 were effective for protoplast formation. Dependence of the efficiency of protoplast formation on the culture period was examined for Lentinus edodes, Pholiota nameko, Grifola frondosa and Auricularia polytricha. The efficiency was also found to be dependent on the strain of the same mushroom. In the course of the study, the enzyme system containing Novozym 234 and chitinase was found to be highly efficient for protoplast formation from mycelia of mushrooms of the genus Pleurotus. Using the strain, FMC 235, of Pleurotus ostreatus, the authors studied conditions for protoplast formation using this enzyme system, these are, ph of the enzyme solution, effect of osmotic stabilizers, time course of protoplast formation and the effect of media used for mycelial culture. Protoplast of Lentinus edodes and Pleurotus ostreatus could be cultured to form mycelia, and culture conditions were further studied. In the course of the study, the authors found that by starting from dikaryotic mycelia of Pleurotus ostreatus, they could get monokaryotic mycelia as well as dikaryotic mycelia after formation of protoplast from dikaryotic mycelia and culture. The authors conclude that this is a simple method of monokaryotization of dikaryotic mycelia. Introduction Protoplast is a cell whose envelope lacks a cell wall and is composed only of plasma membrane. Protoplasts have been used in cell fusion of plants and fungi for their breedingm>. In addition to cell fusion, protoplasts are useful in such aspects of genetics and breeding of microorganisms as transformations> and artificial mutation4>5>. Protoplasts are becoming more and more important also in studies of physiology of fungi as well as in genetics and breedings>. In recent years protoplasts have also been studied for mushrooms7>9>. But for main cultivated mushrooms in Japan such as Lentinus edodes (Berk.) Sing., Pholiota nameko (T. Ito) S. Ito et Imai, Grifola frondosa S. F. Gray and Pleurotus ostreatus (Fr.) Que!., protoplasts have not been studied sufficiently. In this report we studied formation of protoplasts from mycelia of 12 edible mushrooms including main cultivated mushrooms mentioned above, and culture of Pleurotus ostreatus and Lentinus edodes. elsew herelol. A part of this paper was presented Received August. 9, 1986 { i\i-47 Forest Protection-47 (1) (2) (3) (4) (5) Forest Protection Division

2 -156- Materials and Methods Origanisms Strains of 12 mushrooms, that is Lentinus edodes, Pleurotus ostreatus, Flammulina velutipes (Fr.) Sing., Pholiota nameko, Grifola frondosa, Pleurotus cornucopiae (Pers.) Rolland, Hypsizygus marmoreus (PK.) Bigelow, Tricholoma matsutake Ito et Imai, Auricularia polytricha (Mont.) Sacc., Agaricus bisporus (Lange) Sing., Pleurotus salmoneo-stramineus Vassilieva, Pleurotus cystidiosus. K. Miller, and two Pleurotus spp. were used. All the strains were subcultured from stock cultures of Section of Mushroom of this institute (strains with FMC number) and were maintained on potato-dextrose-agar (PDA) medium. Monokaryotic mycelia obtained by single spore isolation from fruiting bodies of these stock cultures were also used. Prepararion of protoplasts from mycelia Forty ml of a liquid media in a 1 ml Erlenmeyer flask containing sucrose (1%), malt extracts (1%) and yeast extracts (. 4%) (SMY medium) were inoculated with the mushroom mycelia used in the experiment. After culturing at 25 C for 1-2 days depending on the kind of mushroom, mycelia were fragmented in a homogenizer (Nippon Seiki Co., Ltd.) in SMY medium at 12 rpm for the time depending the kind of mushrooms. Except for the examination of media, 5 ml of the medium containing the fragmented mycelia were re-inoculated to 4 ml of fresh SMY medium and cultured for 2-7 days at 25 C. Mycelia were filtered by a nylonmesh and washed with a buffer solution containing. 5 M mannitol,. 5 M maleic acid-naoh, ph 5. 5 (buffer A). Mycelia were resuspended in 3 ml of buffer A containing various combinations of enzymes listed in Table 1, and incubated in a water bath at 25 C-3 C with reciprocal shaking (6 revolutions/min.). After incubation, remaining mycelia were removed by filtration through Mylacloth (Calbiochem. Co., Ltd.). For tests of preparation of protoplasts, a number of protoplasts present in the filtered solution were counted by a haemocytometer (Thoma Table 1. Mycolytic enzymes used in this study. Enzyme Source Supplier /Manufacturer Cellulase "Onozuka" R-IO* Trichoderma viride Y akult Pharmaceutical lndust. Co. Ltd. Cellulase "Onozuka" RS* Trichoderma viride Yakult Pharmaceutical Indust. Co. Ltd. Chitinase Streptomyces griseus Sigma Chemical Co. Driselase Irpex lacteus Kyowa Hakko Kogyo Co. Ltd. {3-G 1 ucronidase Helix pomatia Sigma Chemical Co. Macerozyme R-1 Rhizopus sp. Y akult Pharmaceutical Indust. Co. Ltd. Novozym 234 Trichoderma harzianum Novo Industri A/S Enzyme Division Zymolyase 5 Arthrobacter luteus Kirin Breweries Co. Ltd. * Cellulase "Onozuka" R-1 and Cellulase "Onozuka" RS are abbreviated as "Cellulase R-1" and "Cellulase RS," respectively, in this paper.

3 -157- counting chamber). In each test, an average of three replications were shown. For other tests, the filtered solution was centrifuged at 53Xg for five minutes and precipitated protoplasts were resuspended in the buffer A. Protoplasts were purified as follows (when necessary). Protoplasts resuspended in buffer A were layered on the. 7 M sucrose solution containing. 5 M maleic acid-naoh, ph 5, 5, in a centrifuge tube and centrifuged at 53Xg for five minutes. Protoplasts floating on the upper surface of the sucrose layer were recovered and resuspended in. 7 M mannitol containing. 5 M maleic acid-naoh, ph Culture of protoplasts Protoplasts were prepared aseptically, washed once with buffer A and serially diluted with the same buffer. The protoplast solution (. 1 ml) was mixed with 1 ml of SMY or GMY medium containing the osmotic stabilizer, additives, and agar or agarose, and cultured in 9 em plastic Petri dishes sealed with parafilm, at 25"C. For the test of the effect of osmotic stabilizers,. 5 M sucrose was replaced with other osmotic stabilizers. For liquid cultures,. 1 ml of protoplast solution was mixed with the SMY or GMY medium containing the osmotic stabilizer and additive but without agar. Esterase isozymes Esterase isozymes of mycelia of Pleurotus ostreatus cultured in the SMY medium for three weeks were separated and detected as described previously 11 >. Enzymes Enzymes used for the preparation of protoplasts in the present study are listed in Table 1. Chemicals Yeast extracts an nutrient broth were obtained from Oxoid Limited Co.. Malt extracts were obtained from Kyokuto Seiyaku Ind. Co., Polypeptone was obtained from Wako Pure Chemical Ind. Ltd., Potato-dextrose-agar was obtained from Nissui Seiyaku Co., Other chemicals were obtained from Wako Pure Chemical Ind. Ltd. or Nakarai Chemicals, Ltd.. Results 1. Isolation of Protoplast& from Mycelia of 12 Mushrooms Efficiency of protoplast formation from mycelia depended firstly on the combination of enzymes used for preparation. Several combinations of commercial.enzymes, except for Novozym 234 and chitinase, listed in Table 1, were tested for the preparation of protoplasts from Lentinus edodes, Pleurotus ostreatus, Plwliota nameko and Auricularia polytricha. Results are shown in Tebles 25. As shown in these tables, one-enzyme systems were not generally effective. In most cases, mixture of two or more commercial enzymes were effective. Enzyme systems containing a) Cellulase RS and 13-Glucronidase or b) Cellulase RS, Driselase and 13-Glucronidase or c) Cellulase RS, Driselase and Zymolyase 5 showed better results. For a combination of enzymes, that is, for one enzyme system, the number of protoplasts formed were dependent on the kind of mushrooms. The dependence of the number of protoplasts formed from cultured mycelia on different strains of the same mushroom, and on the culture period was tested using four strains each of Lentinus edodes, and Plwliota nameko, three strains of Grifola frondosa, and a strain of Auricularia polytricha. Results are shown in Figs. 14. The enzyme system used in this

4 Table 2. Number of protoplasts formed from mycelia of Lentinus edodes (Strain FMC 2) using several combinations of enzymes. Combination of enzymes Driselase 2% Cellulase RS 2% Macerozyme R-IO 2% Zymolyase 5. 2%,s-Glucronidase. I ml/ml Cellulase R-1 2% Driselase 2%+Cellulase RS 2%+Zymolyase 5 o. 2% Driselase 2%+Macerozyme R-IO 2%+Cellulase R-IO 2% Driselase 2% +,g-glucronidase. I ml/ml Cellulase RS 2% +,g-glucronidase o. I ml/ml Driselase 2%+Cellulase RS 2%+13-Glucronidase. I ml/ml Number of protoplasts per g fresh weight. 72X JOG 6.81X16 O.llXIOG.14XJOG. 17XJOG O.OOXJOG X J 6 I. 55 X JOG. 82X JO& X JQ& X JOG Incubations were performed at 25 C for 5 hours. Table 3. Number of protoplasts formed from mycelia of Pleurotus ostreatus (Strain FMC 235) using several combinations of enzymes. Combination of enzymes Driselase I% Cellulase RS I% Macerozyme R-IO I% Zymolyase 5. I%,g-Glucronidase. I ml/2 ml Driselase 2%+Cellulase RS I% Driselase 2%+Zymolyase 5. I% Driselase 2% +,g-glucronidase. I ml/2 rnl Driselase 2% +Cellulase RS I%+ Zymolyase 5. I% Number of protoplasts per g fresh weight.7xjog 17.43XJOG.94XJOG O. 38X 1G 68.13X XJOI 25.3X X J X JOG Incubations were performed at 25 C for 5 hours. Table 4. Number of protoplasts formed from mycelia of Pholiota nameko (Strain FMC 262) using several combinations of enzymes. Combination of enzymes Driselase 2% Cellulase RS 2% Macerozyme 2% Zymolyase 5. 2% Cellulase R-1 2% Driselase 2%+Cellulase RS 2%+Zymolyase 5.2% Driselase 2% + Macellozyme 2% +Cellulase R -1 2% Cellulase RS 2% +,g-glucronidase o. 1 ml/ml Number of protoplasts per g fresh weight. 83X JOG I. 8X JOG. 54 X JOG.71XJOG. 62X X X X!6 Incubations were performed at 25 C for 5 hours.

5 -159- Table 5. Number of protoplasts formed from mycelia of Auricularia polytricha (Strain FMC 356) using several combinations of enzymes. Combination of enzymes Driselase 2% Cellulase RS 2% Cellulase R-IO 2% Driselase 2% +Cellulase RS 2% + Zymolyase 5. 2% Driselase 2%+Macerozyme 2%+Cellulase R-IO 2% Cellulase RS 2% +,B-Glucronidase. I ml/ml Cellulase RS 2% + Zymolyase 5, 2% Number of protoplasts per g fresh weight, oox X 1 6 O.OOX! X1 6 I, 3X X 16 15,13XJ6 Incubations were performed at 25 C for 5 hours. x1" M..<:: "' LL tlo tl c. "'.8 :: t. E ::::J z, I I ' \ X I I ' \ ' / 'I \.,, ' I \ I \ ' ' 'b Fig. 1. Culture Period (days) Effect of culture period on the formation of protoplasts from mycelia of different strains of Lentinus edodes. Incubations were performed at 25 C for 5 hours. FMC 2:-, FMC 8:---, FMC 29: x---x, FMC 45: x-x. '.s;;;.s;;; X 1 <f) l!! U- " A... ' I I' ' \ I \ J<-..., '\ I'... ' Culture Period (days)... '......',, Fig. 2. Effect of culture period on the formation of protoplasts from mycelia of different strains of Pholiota nameko. Incubations were performed at 25 C for 5 hours. FMC 281:--, FMC 282:x---x, FMC 289:---, FMC 295:x---x.

6 -16-.<::: tld X1 6..c: e l.i... tld Fig. 3. Effect of culture period on the formation of pro toplasts from mycelia of different strains of Grifola frondosa. Incubations were perfor med at 25 C for 5 hours. FMC 32:-, FMC32l:x---x, FMC 323: VI c. "' :: 1i ;;;::..::.===.:::..::=.llf-...j--- z Culture Period (days) X1' -& 45...c:: VI e u. Vl tl c. "' :: Vl. E :J z Culture Period (days) Fig. 4. Effect of culture period on the formation of protoplasts from mycelia of a,strain of Auricularia polytricha, FMC 356. Incubations were perfomed at 25 c for 5 hours. experiment was Cellulase RS, 2%, Macerozyme R-1, 2%, Driselase, 2%, and Zymolyase 5,. 2%. As seen in these figures, number of protoplasts is dependent on the strains used for a mushroom. The effect of the culture period was also dependent on the strain. But protoplast formation at 7 days was generally lower than those at 2 days and/or 4 days. Formation of protop!asts from mycelia was also tested for strains of seven mushrooms using enzyme system 1) Cellulase RS, 2%, Driselase, 2%, Macerozyme R-1, 2%, and Zymolyase 5,. 2%, and 2) Cellulase R-1, 2%, Driselase, 2%, Macesozyme, 2%, and Zymolyase 5,. 2%. Results are shown in Table 6. In, some experiments, formation of protoplasts from mycelia of Tricholoma matsutake was ascertained (Fig. 5). We could not find any effective enzyme system for Agaricus bisporus. 2. Highly efficient formation of protoplasts from mycelia of Pleurotus ostreatus. In the process of the present study, a cell wall lytic enzyme, Novozym 234, became available. So, authors tested this enzyme as well as chitinase, which was known to degrade chitin, an important component of the cell wall of fungi. Results of the test for the prepa ration of protoplasts from mycelia of a strain of Pleurotus ostreatus (FMC 235) is shown in

7 -161- Table 6. Number of protoplasts formed from mycelia of several mushrooms using two enzyme systems. Mushroom and strain Pleurotus ostreatus FMC 235 FMC 236 FMC 237 FMC 238 FMC 239 FMC 24 FMC 241 FMC 242 FMC 243 FMC 244 FMC 245 FMC 246 Pleurotus cystidiosus FMC 256 Pleurotus salmoneo-stramineus FMC 252 Pleurotus sp. FMC 253 Pleurotus cornucopiae FMC 257 Hypsizygus marmoreus FMC 228 FMC 229 FMC 231 FMC 232 FMC 233 Flammulina veltipes FMC 223 FMC 224 Number of protoplasts per g fresh weight Enzyme system I) Enzyme system 2) 131 X J6 29X X 16?OX X 16 14X X 16 22X X!6 26X X!6 26X1 6 23X 16!9X1 6!39X X X X1S 181 X16!29X X16 78X 1 6!29Xl6 36X X X X X X 16 16Xl 6 16Xl6 6X X1 6 68X1S 7X X1S 23X1 6 4X 1 6!65X16 64Xl 6 17 X X1 6 78X 16 45X X X 1 6 Incubations were performed at 25 C for 5 hours. Enzyme system 1) Cellulase RS 2%, Driselase 2%, Macerozyme R-1 2% and Zymolyase 5,2% Enzyme system 2) Cellulase R-1 2%, Driselase 2%, Macerozyme R-1 2% and Zymolyase 5. 2% Fig. 5. Formation of protoplasts from mycelia of Tricholoma matsutake. A : protruding protoplasm of mycelia formed in the process of formation of protoplasts. B : protoplast released from mycelium,

8 -162- Fig. 6. Enzyme systems containing Novozym 234 showed better results and produced a high number of protoplasts even at 2 hours incubation. Further tests were conducted on enzyme system E (Novozym 234, 1%, chitinase,.1%). Protoplasts formed by these enzymes from mycelia of Pleurotus ostreatus are shown in Fig. 7. Because this enzyme system was very effective, authors further studied the various con ditions for protoplast formation from mycelia of Pleurotus ostreatus. Firstly, ph dependence of protoplast formation was studied. Results are shown in Fig. 8. High yield was obtained between ph and further experiments were performed at ph The effect of osmotic stabilizers on the protoplast formation is shown in Fig. 9. In further experiments, authors used mannitol routinely. Fig. 1 shows the time course of protoplast formation for mycelia grown at 3 C and 15 C. Mycelia grown at 3 C produced a higher number of protoplasts in a shorter incubation time than from mycelia grown at l5 C. But mycelia grown at 15 C produced a higher number of protoplasts at 3 hours. The number of pro toplasts formed from both mycelia grown at 15 C and those grown at 3 C decreased at 5 hours..<:.<:: V1!!! LJ ,_ "" 2 V1 a. "' :.D E "' z X r """ 2. - r r- - r- -. _ A B c D E F G Combination of Enzymes Fig. 6. Formation of protoplasts from mycelia of Pleurotus ostreatus (FMC 235) using several combinations of enzymes. A : Cellulase RS 2% + Driselase 2% + Zymolyase 5. 1%. B :,8-Giucronidase. 1 ml/ml. C:,8-Glucronidase.1 ml/ml+cellulase RS 1%. D : Novozym 234 1%. E: Novozym 234 1%+Chitinase.1%, F: Novozym 234 1%+,8-Glucronidase.5ml/ml. G: Novozym 234 1%+Cellulase RS 1%. For each combination of enzymes, incubations were performed at 3 C for 2 hours (shown by left bar) and 4 hours (shown by right bar).

9 -163- Fig. 7. Protoplasts of Pleurotus ostreatus prepared from mycelia using Novozym 234 and chitinase. 2 en a. "'.8 a:... Q).. E :J z Q).;::; > Qi "' :: 7.5 ph of the Enzyme Solution t: "' 1. r-. "' e """" "- r- n E :> 5.,_ z "' >...- r- a; :. o:s: ':3g; -x -< o:s: :> (jlg co 6 (jl[ '->Q "'" (]1 2[?,: 2" 2f' "'"' " (]1-2 2 (\) Osmotic Stabilizer Fig. 8. PH dependance of protoplast formation from mycelia of Pleurotus ostreatus (FMC 235). Incubations were performed at 3 C for 2 hours. Fig. 9. Effect of osmotic stabilizers on the protoplast formation from mycelia of Pleurotus ostreatus (FMC 235). Incubations were performed at 3 C for 2 hours. Efficiency of protoplast formation was found to be dependent on the medium used for culture of mycelia. Effect of media used for preparation of mycelia is shown in Table 7. Using the method described here, we could prepare protoplasts very efficiently from mycelia of Pleurotus ostreatus, P. salmoneo-stramineus, P. cystidiosus and two Pleurotus spp. 3. Culture of protoplasts

10 -164- X1 7..c: 3. <I) t) c. "' b : 15. E z " Period of Incubation (hours) Fig. 1. Time course of protoplast formation from mycelia of Pleurotus ostreatus (FMC 235) grown at 3 C and l5 C. Incubations were performed at 3 C. for 2 hours. : Mycelia grown at 3 C. : Mycelia grown at 15 C. Table 7. Effect of the culture medium on the number of protoplasts formed from mycelia of Pleurotus ostreatus (FMC 235) grown in the medium. Medium MM + 1% Casamino acids MM+t% Polypeptone MM+l% Nutrient broth SMY t% Sucrose+ 1% Polypeptone+O. 4% Yeast extracts 1% Casamino acids+ I% Malt extracts+o. 4% Yeast extracts 1% Polypeptone+ 1% Malt extracts+o. 4% Yeast extracts 1% Nutrient broth+ 1% Malt extracts+ a. 4% Yeast extracts 1% Malt extracts+o. 4% Yeast extracts No. of protoplasts per g fresh weight,5xj7 14, 3X! X!7 2, ox XI7 26, I XJ7 31, 9X! X!7 44.6XJ7 Incubations were porformed at 3 C for 2 hours. MM : Diammonium Hydrogenphosphate 1. 5 g Glucose 2g Potassium Dihydrogen Phosphate. 45 g di-potassium Hydrogen Phosphate 1 g Magnesium Sulfate. 5 g Thiamine Hydrochloride 12 pg Distilled water 11 Protoplasts could be cultured both in liquid media and in solid media. Protoplasts prepared from mycelia of Pleurotus ostreatus (FMC 235) and Lentinus edodes (FMC 2) using the enzyme system Cellulase RS. 2%, Driselase, 2%, Zymolyase 5,. 2%, were first cultured in the liquid media and then in solid media. Results are shown in Table 8 and Table 9. As

11 -165- Table 8. Culture of protoplasts obtained from mycelia of Pleurotus ostreatus (FMC 235)* Osmotic stabilizers and additives No. of colonies for protoplasts cultured in liquid media for* 2 hours I I day 2 days I 3 days I 4 days Mannitol. 5 M 7 1I MgS 4.5M KCl.3M I5 7 Saccharose. 5 M 3 I MgS 4. 5 M+O.I% NAG** 7 I7 55 I1 - MgS 4. 5 M + 1% Polypeptone * Protoplasts prepared using the enzyme system Cellulase RS 2% + Driselase 2% + Zymolyase 5. 2%, were first cultured at 25 C for 2 hours-4 days in liquid media containing glucose 1%, malt extracts 1%, yeast extracts. 4% (GMY) and osmotic stabilizers and additives shown in the left column and then cultured at 25 C in solid media containing GMY,. 6 M mannitol and. 5% Agar. Number of colonies thus formed after 2 weeks of total culture period were counted. ** NAG: N-acetylglucosamine. Table 9. Culture of protoplasts obtained from mycelia of Lentinus edodes (FMC 2)* Osmotic stabilizers and additives No. of colonies for protoplasts cultured in liquid media for* 2 hours I 1 day 2 days 4 days 7 days Mannitol. 5 M MgS 4.5M KCl.3M Saccharose o. 5 M MgSO,.5M+I% NAG** MgS 4. 5 M +I% Polypeptone *, ** ; See legends for Table I II 2I I3 83 4I I IO Fig. 11. Regeneration of a protoplast of Pleurotus ostreatus.

12 -166- seen in these tables, first culture in liquid media improved the efficiency of culture of protoplasts for both mushrooms. But the effect of osmotic stabilizers and additives were different for different mushrooms. Because N-acetylglucosamine improved the culture of protoplasts of Pleurotus ostreatus, it was added to media in the following experiments. Examples of regenerating protoplasts and hyphae regenerated from protoplasts of Pleurotus ostreatus in liquid media are shown in Figs. 11 and 12. For protoplasts of Pleurotus ostreatus prepared using the enzyme system, Novozym 234+ chitinase, the effect of preculture in liquid media was not so clear. The effect of osmotic stabilizers, however, was clearly observed. An example of the effect the above on the culture of protoplasts of Pleurotus ostreatus in solid media is shown in Fig. 13. For Pleurotus ostreatus, MgS 4 was effective. In the culture of protoplasts, ph of the medium had a serious effect on the culture Fig. 12. Hyphae of Pleurotus ostreatus regenerated from a protoplast. 1 Fig. 13. Effect of osmotic stabilizers on the culture of protoplasts of Pleurotus ostreatus (FMC 235). Protoplasts were prepared from mycelia of FMC 235 using Novozym 234 1%+chitinase.1%. They were washed and properly diluted in. 5 M mannitol and then cultured at 25 C in solid media containing SMY, osmotic stabilizer,.1% N-acetylglucosamine and. 7% agarose. After 7 days, numbers of colonies were counted. <J) til c: u >- til. E z " 5 c;s: -(f) as: -x -?< -(f) ) On Oo <. Ol (Jl(f) Olo (Jl:J (Jl N (na- <f> -::;- 1? ;:::, ;::: '. CD [ - -Q. <f> CD Osmotic Stabilizers

13 VI Q) c: <..) Q). E ::J z 1 ph of the Medium Fig. 14. Effect of ph of the medium on the culture of protoplasts of Pleurotus ostreatus (FMC 235). For culture of protoplasts, see the legend of Fig. 13. I I I t t Fig. 15. Monokaryotization of dikaryotic mycelia of Pleurotus ostreatus, FMC 235, by formation and culture of protoplasts. Esterase isozymes of mycelia which were obtained by culture of protoplasts prepared from dikaryotic mycelia of FMC 235, are shown here. An arrow of thick, solid line shows isozymes of the dikaryotic mycelia and arrows of narrow, solid and broken line show isozymes of two types of monokaryons. efficiency. Protoplasts of FMC 235 were prepared and diluted as described in the legend of Fig. 13 and cultured in media containing SMY,. 5 M MgS 4, and maleic acid-naoh buffer to adjust ph. shown in Fig. 14. After 9 days of culture, the number of colonies were counted. Results are As seen in this figure, ph near neutrality is good for the culture of pro toplasts of Pleurotus ostreatus in the ph range tested here. In the course of experiments of preparation and culture of protoplasts of Pleurotus ostreatus, we found that monokaryotic mycelia could also be obtained from the culture of protoplasts. Colonies whose hyphae have no clamps could be recovered. They could be mated with other monokaryotic.mycelia of other strains of Pleurotus ostreatus to produce dikaryotic mycelia. Esterase isozymes were analyzed for these monokaryons, cultured from protoplasts, as well as for dikaryons, cultured from protoplasts, and original stock. The result is shown in Fig. 15. As seen from the figure, we could get two types of monokaryons. Discussion We were able to prepare protoplasts from mycelia of 12 species of mushrooms. Although Moores> prepared protoplasts from Coprinus cinereus using only chitinase or Helicase and de

14 -168- Vries et azm prepared protoplasts from a number of mushroom using only an enzyme prepared from Trichoderma viride, our results indicated that generally mixed enzyme systems were more effective. This fact seems to be related to the complex nature of the cell wall of mushroom mycelials>. We could find effective enzyme systms for important cultivated mushrooms, Lentinus edodes, Pleurotus ostreatus, Pholiota nameko and Auricularia polytricha. Also for Gri[ora [rondosa we could get a fairly high number of protoplasts. Figs. 1-4 and Table 6 clearly shows that the efficiency of an enzyme system is highly dependent on the strain, even for the same mushroom. It is also dependent on the culture period of mycelia. As shown in Figs. 1-4, seven-day cultures were generally ineffective for protoplast formation compared with younger cultures. Table 7 shows protoplast formation from Pleurotus ostreatus is also affected by the media used for culture of mycelia. It was also observed that cultures containing large fractions of aerial hyphae were ineffective for protoplast formation. These facts indicate that protoplast foramation from mycelia is severely controlled by the physiological nature of mycelia. We have established highly efficient enzyme systems for Pleurotus ostreatus as shown in Fig. 6. The system seem to be superior to the method described by Yamada et el (1983)14> for the preparation of protoplasts from mycelia of Pleurotus ostreatus. One of our enzyme systems, Novozym 234+Chitinase, was effective not only for Pleurotus ostreatus but also for other mushrooms of the genus Pleurotus. Protoplasts of Pleurotus ostreatus and Lentinus edodes could be regenerated and cultivated. Materials used for osmotic stabilizers had a serious effect on the efficiency of regeneration and cultivation of protoplasts. But as seen in Table 8, 9 and Fig. 13, an effective osmotic stabilizer was different for P. ostreatus and L. edodes. Regeneration of protoplasts of P. ostreatus were more effective at ph 6. 5 than at more acidic ph regions. As described in "Results", dikaryotic mycelia of Pleurotus ostreatus could be easily monokaryotized by protoplast formation. Two types of monokaryon could be obtained as was evidenced by isozyme analysis. This technique seems to be more effective and easier than monokaryotization by culture in the special medium 1 5l, Monokaryotization by protoplast formation seems to have two advantages over the conventional method of getting monokaryotic mycelia from isolated basidiospores. 1. We can get monokaryons easily and at any time. 2. We can always get monokaryons with a constant genetic nature in breeding. Therefore monokaryotization by protoplast will be very useful in the practical breeding of mushrooms. References 1) CaALEFF, R. S.: Genetics of Higher Plants, Applications of Cell Culture, Cambridge University Press, 184 P. P., (1981) 2) FERENczv, L. : Microbial Protoplast Fusion. In "Genetics as a Tool in Microbiology" ed. by S. W. Glover and D. A. Hopwood. Cambridge University Press., p. 1-p. 34, (1981) 3) SANDRI GoLoiN, R. M., A. L. GoLDIN, M. LEVINE and J, GLoRIOso: High-Efficiency Transfer of DNA into Eukaryotic Cells by Protoplast Fusion. Methods in Enzymology, Academic Press, 11, p. 42-p. 411, (1983) 4) KELLER, U. : Highly Efficient Mutagenesis of Claviceps purpurea by Using Protoplasts.

15 -169- Appl. Environ. Microbial., 46, , (1983) 5) KIM, K. S., N.Y. CHo, H. S. PAI and D. D. Y. RYu: Mutagenesis of Micromonospora rosaria by Using Protoplasts and Mycelial Fragments. Appl. Environ. Microbial., 46, , (1983) 6) VILLANUEVA, J, R., I. GARCIA AcHA, S. GAscoN and F. URusuRu: Yeast, Mould ane Plant Pro toplasts, Academic Press, 381 pp., (1973) 7) de VRIES,. M. H. and }. G. H. WEssELs: Release of Protoplasts from Schizophyllum com mune by a Lytic Enzyme Preparation from Trichoderma viriede. J. Gen. Microbial., 73, 13-22, (1972) 8) MooRE, D. : Production of Coprinus Protoplasts by Use of Chitinase or Helicase. Trans. Br. mycol Soc., 65, , (1975) 9) UsHIYAMA, R. and Y. NAKAI : Protoplasts of Shiitake, Lentinus edodes (Berk.) Sing Rept. Tottori Mycol. Inst. (Japan), 15, 1-5, (1977) 1) HMASA, M., Y. AsE, H. FuRUKAwA, M. TANIGUCHI and H. NEDA: Preparation, Culture and Fusion of Protoplasts of Mushrooms of the Genus Pleurotus. In "proceedings of the 28th annual meeting of the Mycological Society of Japan", p. 59, (1984) 11) HMASA, M. and H. FURUKAWA :Analysis of esterase and malate dehydrogenase isozymes of Lentinus edodes by isoelectric focusing for the identification and discrimination of stocks. Trans. mycol. Soc. Japan, 27, 79-9, (1986) 12) de VRIES,. M. H. and J, G. H. WEsSELs: Effectiveness of a Lytic Enzyme Preparation from Trichoderma viride in Releasing Spheroplasts from Fungi, Particularly Basidiomycetes. Antonie van Leeuwenhoek, 39, 397-4, (1973) 13) FARKAs, V. :The Fungal Cell Wall. In "Fungal Protoplasts: Applications in Biochemistry and Genetics" ed. by J. F. Peberdy and L. Ferenczy, Marcel Dekker, Inc., p. 3-29, (1985) 14) YAMADA,., Y. MAGAI, Y. KAsHIWAGI, T. SHIRATORI and T. SAsAKI: Formation and Regene ration of Flammulina velutipes and Pleurotus ostreatus Protoplasts. Nippon Shokuhin Kogyo Gakkaishi, 3, 495-5, (1983) 15) LEAL-LARA, H. and G. EGER HUMMEL: A Monokaryotization Method and its Use for Genetic Studies in Wood-rotting Basidiomycetes. Theor. Appl. Genet., 61, 65-68, (1982)

16 林業試験場研究報告第 343 号 日本産栽培きのこのプロトプラストの調製と培養 大政正武 ω. 阿部恭久叩 古川久彦 別 谷口実 引 根田仁 (6) 摘要 プロトプラストは, 細胞融合や形質転換をはじめとする, きのとではとれまで行われていなかった新しい育種法を可能にするものとして, 大きな期待が寄せられている プロトプラストの作成と菌糸の再生についてはとれまでスエヒロタケやヒトヨタケ属のきの ζ などについての研究はあるがが, シイタケ, ヒラタケ, ナメコ, マイタケなど日本産の栽培きの乙については研究が少なかった 本研究は, 乙れらの日本産の栽培きのとについて, プロトプラストの調製法と培養法を検討し, 上記の新しい育種法を開拓するための基礎的知見を得るととを目的として行ったものである 日本産栽培きのと 12 種の菌糸からプロトプラストの調製を行った とれらにはシイタケ, ヒラタケ, エノキタケ, ナメコ, マイタケ, マツタケ他が含まれる きのとの菌糸を液体培養し, 集菌後, 菌の細胞壁溶解酵素の処理でプロトプラストを得た 市販酵素の組み合せのうち, a) セ Jレラーゼ RS, ß- クツレクロニダーゼ, b) セルラーゼ RS, ドリセラーゼ, β ーグルクロニダーゼ, c) セ Jレラーゼ RS, ドリセラーゼ, ザイモリアーゼ 5 の酵素系が良い成績を示した シイタケ, ナメコ, マイタケ, アラゲキクラゲにつ いてプロトラプスト調製に対する菌糸の培養期間依存性を検討した また系統の違いによってもプロトプ ラスト調製は異なった ノポザイム 234 とキチナーゼの組み合せはヒラタケ属のきのとに対してきわめて 良い成績を示したので, プロトプラスト調裂のさらに詳しい条件, すなわち, ph 依存性, 浸透圧調節剤 の影響, 処理時間依存性, 菌糸培養の培地の影響等を検討した シイタケとヒラタケのプロトプラストは 培養を行なうととができたので, その条件をさらに検討した その過程で, ヒラタケの 2 核菌糸からプロ トプラストを得て培養すると 2 核菌糸と同時に 1 核菌糸が再生するととが見出され, 2 核菌糸の 1 核化の 簡便法として使えるととが分った 1986 年 8 月 9 日受理 (5) 保護部